qPCR common problems and solutions

How to judge the validity of qPCR data

Usually it is necessary to combine amplification curve, melting curve, and standard curve for comprehensive analysis.

1. The amplification curve should present a smooth S-shaped curve , with no amplification at the beginning, the peak time is normal, and it can reach the plateau. The Ct value obtained from the amplification curve is an important reference for judging the success of the experiment. The Ct value has a certain credible range. It is generally recommended that the positive result (scientific research) be adjusted to between 15-30, the negative control Ct>35, and the internal reference Genes are generally less than 20, and the difference in Ct value of the internal reference between samples does not exceed 1 , indicating that the expression of the internal reference gene is stable, and the standard deviation of the Ct value of duplicate wells does not exceed 0.5 .
2. The melting curve should present a single peak with a small width indicating specific amplification.
3. The slope of the standard curve ranges from -3.58 to -3.10 , and the amplification efficiency of the corresponding primers is 90% to 110% . R ² represents the degree of linear fit, which is generally greater than 0.98 .
   

qPCR FAQs and Solutions

1. The amplification curve is unstable

• Possible reasons: RNA purity is low; there are many impurities in the system; the instrument has been used for too long.
• solution:
  1. Extract high-purity RNA and handle it carefully;
  2. Calibrate the instrument.

2. Amplification cannot reach plateau

• Possible reasons: the template concentration is too low; the number of cycles is too small; the reagent amplification efficiency is low.
• solution:
  1. Increase the amount of templates;
  2. Increase the number of cycles;
  3. Use the standard curve to determine the amplification efficiency, increase the concentration of magnesium ions, and use reagents with high amplification efficiency.

3. Fluorescence drop

• Possible reasons: Degradation exists; template concentration is too high.
• solution:
  1. Improve system purity;
  2. Reduce the amount of templates;
  3. Lower the fluorescence threshold.

4. Single peak, not sharp peak

• Possible reasons: related to reagent components; or non-specific amplification of similar size.
• solution:
  1. A temperature span not higher than 7 degrees is considered a usable result;
  2. Perform high-concentration agarose electrophoresis to confirm whether it is a single band.

5. Single peak, Tm below 80 degrees

• Possible reasons: only primer dimers, no target bands ; or the amplified fragment is too short .
• solution:
  1. Check whether to join the template;
  2. Perform agarose electrophoresis to determine whether the band size is correct.

6. Double peak, lower peak Tm before 80 degrees

• Possible cause: Excessive primers form primer-dimers due to low template concentration or high primer concentration.
• solution:
  1. Appropriately increase the annealing temperature;
  2. Increase the amount of template and reduce the concentration of primers;
  3. Redesign primers.

7. Severe double peaks, Tm are all after 80 degrees

• Possible reasons: poor primer specificity or cross-contamination.
• solution:
  1. BLAST checks primer specificity and redesigns primers;
  2. In an ultra-clean bench, use a pipette to add primers separately to avoid cross-contamination.

8. Other abnormal melting curves

• Possible reasons: contamination of the reaction system, failure of reagents; mismatch between consumables and the instrument, incorrect detection channels; the instrument has not been corrected for a long time.
• solution:
  1. Prepare the system in a template-free clean area;
  2. Avoid exposing reagents to strong light or high temperature;
  3. Avoid reagent expiration;
  4. Make positive and negative controls;
  5. Select consumables that match the instrument;
  6. Regularly invite engineers to calibrate the instrument.

9. The linearity of the standard curve is not good

• Possible reasons: sampling error; degradation of standard; high concentration of template or inhibitor; poor primer or probe; low sensitivity and decreased activity of PCR enzyme.
• solution:
  1. The standard sample volume is greater than 2ul, the primers are premixed and then divided into multiple wells, the pipette is calibrated regularly, the siliconized pipette tip is selected as much as possible, and the library diluent is used to dilute the standard product to reduce the adsorption of DNA on the consumable tube wall;
  2. Avoid repeated freezing and thawing, and re-prepare diluted standards after degradation is found in electrophoresis detection;
  3. Change the dilution precision and increase the dilution gradient;
  4. Redesign of primers and probes;
  5. Replace the reagent with higher sensitivity and better linearity, calibrate the -20°C refrigerator, and place the enzyme on ice when in use.